Completion of meiosis in male zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) despite lack of DNA mismatch repair gene <Emphasis Type="Italic">mlh1</Emphasis>

Mlh1 is a member of DNA mismatch repair (MMR) machinery and is also essential for the stabilization of crossovers during the first meiotic division. Recently, we have shown that zebrafish mlh1 mutant males are completely infertile because of a block in metaphase I, whereas females are fertile but have aneuploid progeny. When studying fertility in males in a two-fold more inbred background, we have however observed low numbers of fertilized eggs (approximately 0.4%). Histological examination of the testis has revealed that all spermatogenic stages prior to spermatids (spermatogonia, primary spermatocytes, and secondary spermatocytes) are significantly increased in the mutant, whereas the total weight of spermatids and spermatozoa is highly decreased (1.8 mg in wild-type vs. 0.1 mg in mutants), a result clearly different from our previous study in which outbred males lack secondary spermatocytes or postmeiotic cells. Thus, a delay of both meiotic divisions occurs rather than complete arrest during meiosis I in these males. Eggs fertilized with mutant sperm develop as malformed embryos and are aneuploid making this male phenotype much more similar to that previously described in the mutant females. Therefore, crossovers are still essential for proper meiosis, but meiotic cell divisions can progress without it, suggesting that this mutant is a suitable model for studying the cellular mechanisms of completing meiosis without crossover stabilization. Marcelo C. Leal and Harma Feitsma contributed equally to this work. This work was supported by the Brazilian Foundation CAPES, the Cancer Genomics Center (Nationaal Regie Orgaan Genomics), the European Union-funded FP6 Integrated Project ZF-MODELS, and Utrecht University.     

Role of C chemokine lymphotactin in mediating recruitment of antigen-specific CD62L(lo) cells in vitro and in vivo.

In this study we investigated whether T cells expressing high or low levels of CD62L were differentially susceptible to the T cell chemokine lymphotactin. We found that lymphotactin induced preferential migration of antigen-specific (CD62L(lo)) T cells over the nonspecific (CD62L(hi)) T cells in vitro and in vivo. The differing migratory abilities correlated with higher levels of mRNA encoding the lymphotactin receptor (XCR1) on the CD62L(lo) cells compared to the CD62L(hi) cells. Thus, we have identified a coupling mechanism between the activation of T cells and acquisition of new homing properties, in this case conferred by XCR1 expression. These data confirm that at least one function of lymphotactin includes mediating the recruitment of recently activated antigen-specific T cells.     

A novel scheme to assess factors involved in the reproducibility of DNA-microarray data

Background  

In research laboratories using DNA-microarrays, usually a number of researchers perform experiments, each generating possible sources of error. There is a need for a quick and robust method to assess data quality and sources of errors in DNA-microarray experiments. To this end, a novel and cost-effective validation scheme was devised, implemented, and employed.     

Identification and characterization of two novel clostridial bacteriocins,circularin A and closticin 574

Two novel antibacterial peptides of clostridial species were purified, N-terminally sequenced, and characterized. Moreover, their structural genes were identified. Closticin 574 is an 82-amino-acid bacteriocin produced by Clostridium tyrobutyricum ADRIAT 932. The supernatant of the producing strain showed a high level of activity against the indicator strain C. tyrobutyricum. The protein is synthesized as a preproprotein that is possibly secreted via the general secretion pathway, after which it is hydrolyzed at an Asp-Pro site. Circularin A is produced by Clostridium beijerinckii ATCC 25752 as a prepeptide of 72 amino acids. Cleavage of the prepeptide between the third leucine and fourth valine residues followed by a head-to-tail ligation between the N and C termini creates a circular antimicrobial peptide of 69 amino acids. The unusually small circularin A leader peptide of three amino acids is cleaved off in this process. The supernatant of C. beijerinckii ATCC 25752 showed a broad antibacterial activity range.     

Flexible work hours, health and well-being in the European Union: preliminary data from a SALTSA project

Demand for flexible work hours (FWH) is increasing in Europe aimed at increasing the number of production hours on one hand, and, on the other, reducing individual working hours and/or increasing autonomy and control on them. In view of the lack of knowledge of the effects of FWH on health and safety, we started a pilot project, funded by the Joint Programme for Working Life Research in Europe (SALTSA), aimed at: a) comparing the most relevant national legislation and how the EU Directive 93/104 "concerning certain aspects of working time" has been implemented in the member States; b) reporting prevalence and trend of FWH in Europe according to the three EU Surveys on Working Conditions carried out in the last decade; c) collecting practical examples of innovative FWH; d) evaluating their impact on health and safety in relation to work sectors, job demands, social life, aging and gender. Consequent actions are going to include information and consultancy for pertinent authorities and social parties involved, as well as training programmes for Union officials and similar groups concerning the organisation of FWH according to ergonomic principles.     

Voltage-dependent opening of HCN channels: Facilitation or inhibition by the phytoestrogen, genistein, is determined by the activation status of the cyclic nucleotide gating ring

Investigation of the mechanistic bases and physiological importance of cAMP regulation of HCN channels has exploited an arginine to glutamate mutation in the nucleotide-binding fold, an approach critically dependent on the mutation selectively lowering the channel's nucleotide affinity. In apparent conflict with this, in intact Xenopus oocytes, HCN and HCN-RE channels exhibit qualitatively and quantitatively distinct responses to the tyrosine kinase inhibitor, genistein — the estrogenic isoflavonoid strongly depolarizes the activation mid-point of HCN1-R538E, but not HCN1 channels (+ 9.8 mV ± 0.9 versus + 2.2 mV ± 0.6) and hyperpolarizes gating of HCN2 (− 4.8 mV ± 1.0) but depolarizes gating of HCN2-R591E (+ 13.2 mV ± 2.1). However, excised patch recording, X-ray crystallography and modeling reveal that this is not due to either a fundamental effect of the mutation on channel gating per se or of genistein acting as a mutation-sensitive partial agonist at the cAMP site. Rather, we find that genistein equivalently moves both HCN and HCN-RE channels closer to the open state (rendering the channels inherently easier to open but at a cost of decreasing the coupling energy of cAMP) and that the anomaly reflects a balance of these energetic effects with the isoform-specific inhibition of activation by the nucleotide gating ring and relief of this by endogenous cAMP. These findings have specific implications with regard to findings based on HCN-RE channels and kinase antagonists and general implications with respect to interpretation of drug effects in mutant channel backgrounds.     

Reversible depression of the reticuloendothelial system by liposomes

The effect of various doses of different types (reverse phase evaporation vesicles and small unilamellar vesicles) of intravenously injected liposomes on reticuloendothelial activity, as measured by the blood clearance rate of intravenously injected carbon, was investigated. Also the effect of pretreatment with reverse phase evaporation vesicles on blood clearance and tissue distribution of a second dose of similar vesicles was determined. For all concentrations used reverse phase evaporation vesicles caused reduction in reticuloendothelial activity at least up to 4 h after injection. 24 h after administration the rate of carbon clearance returned to the control level. On the contrary small unilamellar vesicles did not block reticuloendothelial activity. Pretreatment with reverse phase evaporation vesicles (250 μmol/kg) caused an increased blood level and a decreased hepatic uptake of a second dose of the vesicles, injected 1 h after the first dose. This seems to be due to a depression of reticuloendothelial activity and not to a depletion of opsonins. Pretreatment with small unilamellar vesicles (250 μmol/kg) had no significant influence on the tissue distribution of a second dose of vesicles. Our results clearly indicate that reverse phase evaporation vesicles cause a reversible depression of reticuloendothelial activity and this depression seems to be induced by a saturation of reticuloendothelial cells with liposomes.     

An efficient gene deletion procedure for the mushroom-forming basidiomycete Schizophyllum commune

Gene deletion in Schizophyllum commune is hampered by a low incidence of homologous integration. As a consequence, extensive screening is required to identify a transformant with the desired genotype. To alleviate this and to facilitate the assembly of deletion plasmids, vector pDelcas was constructed. This construct has a set of restriction sites, which allows for directional cloning of the flanking sequences at both sides of a nourseothricin resistance cassette. Moreover, it contains a phleomycin resistance cassette elsewhere in the plasmid, which is used to screen for transformants with an ectopic integration of the pDelcas derivative. The use of pDelcas derivatives in combination with an improved PCR screening protocol permitted the efficient identification of S. commune deletion strains. This procedure may also function in other basidiomycetes.